Field of the Invention
The present invention is generally directed toward an electrochemical method for measuring the activity of proteases using nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). Short peptide substrates specific to disease-related enzymes are covalently attached to the exposed VACNF tips of the NEAs. A redox moiety, such as ferrocene (Fc), can be linked at the distal end of the CNFs. Upon being exposed to a biological sample containing one or more target enzymes, the change in redox signal of the redox moiety due to cleavage of the peptides can be measured using AC voltammetry (ACV) thereby indicating the presence of a specific target enzyme in the sample.
Description of the Prior Art
Globally, it was estimated that about 7.6 million people died in 2007 from various types of cancer, translating into about 20,000 people that die every day. Early diagnosis via cost-effective screens on the high-risk subpopulation is the key for saving millions of lives. Early diagnosis and objective monitoring of treatment response are key components in cancer diagnosis, therapeutic treatment, and integrated management. It has been known that the overexpression of various enzymes such as kinases, phosphatases, and proteases leads to cancers. Cancer metastasis is an uncontrollable cell growth through invasion to neighboring cells and activation of oncogenic pathways. The invasive process particularly involves proteases, a group of enzymes that cause proteolysis to degrade the extracellular matrix components and intercellular cohesive structures of neighboring cells leading to the activation of growth and angiogenic factors. Many clinical therapeutics or drugs under clinical development are based on kinase and protease inhibitors.
Proteases overexpressed in cancer cells and secreted into circulation are not only present as drug targets for the development of protease inhibitors, but also as biomarkers for diagnosis. Three proteases highly expressed in tumors, tumor-associated endothelial cells, tumor-associated macrophages, and other stromal cells are legumain, cathepsin B, and matrix-metalloproteinase 7 (MMP-7). The cysteine protease legumain has a strict specificity for hydrolysis of asparaginyl bonds. It is highly expressed in the metastatic and invasive solid tumors, where it activates other proteases such as cathepsin B, H and L, leading to elastolytic and collagenolytic activities. Although basal proteases are found in normal macrophages, overexpression of these cysteine proteases has not been found in normal tissues. Legumain is found highly expressed in a majority of tumors including carcinomas of the breast, colon, and prostate, and in central nervous system neoplasms. Cathepsin B is overexpressed in various cancers including esophageal adenocarcinoma, breast, colorectal, gastric, lung, and prostate carcinomas. MMP-7 is overexpressed in human cancer tissues and proteolytically degrades extracellular matrix of adjacent cells in cancer progression and invasion. It is recognized as a biomarker for various cancers and is distinct in a number of characteristics from the 23 identified MPPs and over expressed in various cancers including stomach, liver, colon, and breast cancers.
Presently, no commercial legumain- and cathepsin-biosensor have been reported, while diagnostic detection of MMP-7 requires the use of conventional fluorogenic proteolytic beacon and enzyme-linked immunosorbent assay (ELISA). There are three main limitations: 1) only one protease can be analyzed in each measurement, 2) a large amount of sample and reagent is necessary, and 3) a workstation in a centralized lab is required. What is needed is an improved method for rapid detection of early stage cancers and “point-of-care” monitoring of treatment response.